This invention relates to optical detection of analytes, and more particularly to detection of biological, biochemical, and chemical substances using surface plasmon resonance.
Surface plasmon resonance (SPR), the resonant transfer of electromagnetic energy from the evanescent field of a light beam into electron-photon coupled oscillation in some metals, has been used extensively in sensor applications. The magnitude of this resonant phenomenon depends critically on equalizing the phase velocity of excitation beam with that of the surface plasmon wave (SPW), whose propagation velocity strongly depends on the index of refraction of the medium in close proximity to the metal surface. To achieve this equality, the propagation velocity of the electromagnetic wave needs to be reduced. A number of techniques have been used. For example, a conventional SPR spectroscopic device, consisting of a metallic film used with a prism to provide a surface plasmon wave, can be modified by coating the film with a dielectric layer. See, e.g., U.S. Pat. No. 5,991,488.
The most widely deployed technique for SPR detection uses a prism to generate total internal reflection at one of its surfaces. This surface is coated with a thin metallic film, which supports a SPW. See, e.g., U.S. Pat. Nos. 5,991,488; 4,889,427. Changes in the incident light angle, or its wavelength, produce changes in propagation velocity along the prism surface and thus strongly affect the amplitude of the reflected light. The change of the index of refraction at the surface changes the angle at which the resonance occurs. This principle is used in analysis and detection of samples containing analyte. The prism surface is pre-coated with an immobilized layer of a ligand, which has a strong affinity for a analyte, causing the analyte to bind to the surface and thus modify its index of refraction. This modification shifts the SPR curve, i.e., the light intensity versus velocity mismatch, and affects the output light intensity, which is a measure of binding.
Most devices calculate a change in the plasmon resonance angle. See, e.g., U.S. Pat. No. 4,889,427. However, this approach requires a very stable mechanical structure to achieve the requisite sensitivity. The instruments using this approach are large, sensitive to temperature, immobile, and expensive. Furthermore, due to free beam reflection at the surface, it is difficult to expand their operation to more than a few independent sensing channels. Other configurations include the use of waveguides such as optical fibers or planar, single mode structures, designed to detect a shift of the SPR response curve, which measures a change in the index of refraction of the metallic film-abutting layer. See, e.g., U.S. Pat. Nos. 5,815,278; 5,485,277; and 5,359,681. In the case of optical fibers, the wavelength of light can be changed to trace out the SPR response curve.
Single planar waveguides can be used to detect changes in transmitted light intensity, but they lack a free parameter (such as angle) to trace out the response curve. Thus, any spurious change in transmitted light cannot be distinguished from a real signal. Some planar devices include a reference waveguide having a deactivated ligand layer. See U.S. Pat. No. 5,485,277. While this reference controls for mechanical instabilities and nonspecific binding effects but requires a deactivation step and does not account for differences between the deactivated and activated ligand layers.
The invention features a differential mode of detection using surface plasmon resonance (SPR) measurement that avoids mechanical and optical instabilities and enhances detection of analytes including, for example, DNA, antibodies, proteins, and chemical compounds. The invention achieves these results by using sets of optical waveguides having different propagation parameters, or light of different wavelengths, and is suitable for multi-sample and multi-analyte applications in a miniaturized detection system. Furthermore, the invention can include the use of an alternating electric field to reduce nonspecific analyte binding and detection time.
The new detection devices can have one or more sets of two or more waveguides, metallic films (e.g., gold or silver) that support a surface plasmon wave covering at least a portion of each of the waveguides, and ligand layers for binding analytes to the metallic films. The waveguides can be made on a substrate or in optical fibers , wherein the substrate comprises a first material, which can be an optically transparent material (e.g., borosilicate, silicon dioxide or a polymer) with top and bottom surfaces, The top surface is covered by a second material, e.g., magnesium fluoride, having an index of refraction lower than the index of refraction of the first material. The metallic film can cover a portion or the entire length of the waveguides.
In another embodiment of these detection devices, each waveguide in a set on the detection device has a distinct light propagation velocity. To provide this distinct light propagation velocity, each waveguide in a set can have a distinct size or shape (e.g., a distinct width) or be covered by a second material with a distinct thickness.
New methods of differential SPR detection involve transmitting multiple light beams through at least one waveguide on a detection device, where the beams of electromagnetic radiation, i.e., light beams, have different light propagation velocities within the waveguides. The detection device has metallic film that supports a surface plasmon wave and at least in part covers the waveguides. The intensities of the transmitted beams are measured, and a difference between intensities for one or more sets of waveguides are calculated at a first time. A sample, e.g., a liquid or gas, is provided to the metallic film, and a second difference is calculated for the two light beams at a second time. These differences are compared to detect any shift of the SPR curve.
The metallic film can have ligand layers for binding analytes. The two light beams can be light beams with different wavelengths and these light beams can be transmitted through the same waveguide. The two light beams can also be two light beams transmitted through two waveguides having distinct light propagation velocities (i.e., waveguides having distinct shape or size, e.g., distinct widths). Alternatively, where the detection device involves waveguides on a substrate that has a first material with top and bottom surfaces covered by a second material on its top surface that has a lower index of refraction than the first material, the two light beams can be transmitted through two waveguides covered by distinct thicknesses of the second material. The methods can repeat the steps of obtaining a second difference and comparing it to the first difference either continuously or at intervals.
Furthermore, the methods can include providing an alternating polarity electric field to the sample, where the electric field has a field strength less than a binding strength between the ligand layer and the analytes. In another implementation, the methods can involve providing an alternating polarity electric field to the sample, where the electric field has a greater strength during the part of its cycle that causes binding of the analytes than the strength during the part of its cycle that causes unbinding of the analytes.
Detection systems include a cell containing one or more samples that provide the sample to contact at least one metallic film that supports a surface plasmon wave and covers at least a portion of one or more waveguides, at least one light source (i.e., a laser, e.g., semiconductor laser, or laser diode) to transmit light beams into the waveguides, a photodetector to convert the transmitted light into electrical signals, and a processor, e.g. a differential amplifier, to provide results. The processor converts the electrical signals into measured intensities and then computes a first difference between measured intensities for a pair light beams at a first time and a second calculated difference at a later time. Comparing the first difference and the second difference indicates any shift of the SPR curve, and this process can be repeated continuously or at intervals.
The cell can include one or more conduits to flow the sample over ligand layers attached to the metallic films for analyte detection. A differential amplifier or a handheld, mobile, personal, or mainframe computer can be used as a processor. In addition, results can be provided, e.g., in digital, electronic form, to locations physically separated from the sources and detectors by, e.g., satellite, radiofrequency broadcast, fiber optic cable, or electric wire. The detection systems itself can further include a source of voltage connected through wires to physically separated conductive pads, which can include the metallic film, within the cell to provide an electric field across a separation.
A ligand layer is a collection of binding moieties attached to the metallic film either directly or using an intermediary.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The devices, methods, and systems of differential SPR detection offer numerous advantageous in diverse contexts. They can be used to detect a change in a sample property, e.g., a temperature-induced change in viscosity, or a ligand-analyte interaction. They are suitable for laboratory use in both clinical and research settings. In a clinical setting, systems featuring differential SPR detection can enhance efficiency by analyzing a number of samples simultaneously for multiple analytes, with little effort required from the laboratory technician. In a research setting, the ability to study binding kinetics by collecting time-series data is particularly useful. Differential SPR detection can also be used in the home or office, since the devices are easy to use, portable, and inexpensive. The robustness of these systems avoids mechanical and optical instabilities, as well as the lack of human intervention required to maintain them, make them ideal for use in the field. In particular, configuring these SPR systems for communication of results to remote locations is especially attractive as it reduces communication time, transmission errors, and the need for the observer to be physically present at the location where the detection device is being used.